Recipes for reagents formerly made by the student helper
Note: This page is kind of a mess. If you need information on the reagents listed here, please follow the links in their names. YEP Liquid Media (1L) 20 g Bacto Peptone (powder) 10 g Bacto Yeast Extract (powder) 1) Put a little (~100 ml) diH2O in 1L beaker 2) Add powders and stir bar. 3) As it stirs, add diH2O to 960 mL mark (to minimize lumps) 4) When completely clear, pour into bottles (note: only 400 ml can go in a 500 ml bottle) 5) Autoclave 45 minutes (probably less is okay) Sporulation Media (1L) 3 g Potassium Acetate (KOAc or CH3COOK) 0.2 g Raffinose 1) Mix 500 ml diH2O, KOAc, and raffinose in 1000 ml beaker, dissolve 2) Bring volume to one liter with more diH2O 3) Filter sterilize into 4 250 ml bottles 0.5M EDTA (1L) 186.1 g Na2EDTA·2H2O stirring in ~800 ml milliQ-pure H2O in a 1 liter beaker. Add 20 g NaOH pellets, which will allow dissolution of the remainder of the EDTA. Now add additional NaOH, in the form of 50% solution to bring the pH to 8.0 and bring to volume. Autoclave in two 500 ml bottles 10X TE (1L) 10 ml 1M Tris-HCl, pH = 8.00 5 ml 0.2M EDTA (2 ml of 0.5M EDTA) ~1000 ml diH2O Milli-Q 1) Combine Tris-HCl, EDTA, and Milli-Q water in one liter beaker 2) Autoclave or filter sterilize 20% SDS For 4260 ml: Sigma L-5750 diH2O 2L beaker lined w/ plastic bag weigh 852 g SDS. Fill 4L jug part way. Add stir bar. Using funnel, add 850 g SDS. Stir O/N. Continue adding until dissolved. Bring to volume. Aliquot to 1L bottles. No autoclave. Smash ‘n’ Grab Lysis Buffer (80 ml) 0.8 ml 1M Tris pH 8 ( -> 10 mM) 160 µl 0.5M EDTA (-> 1 mM) 1.6 ml 5M NaCl (-> 0.1M) 8 ml Triton X100 20% (->2%) 4 ml 20% SDS (-> 1%) 65.2 ml diH2O No need to be autoclaved. Place 2 x 40 ml into Falcon tubes 1M LiOAc 20.4 g LiAc 10.2 g H2O to 200 ml Milli Q H2O to 100 ml Autoclave (I, too am confused be this recipe as I am transcribing it – Brett) 50% PEG 1) Place 50 g polyethylene glycol powder in beaker 2) Add 30-40 ml diH2O 3) Heat and stir until totally clear. (It takes forever) 4) Dilute to 100 ml with diH2O (wait about 40 min & till gets clear) 5) Transfer to a 125 ml bottle using a steriflip vacuum filter Note: polyethylene glycol comes in a big tub 50% Glucose (400 ml, in 2 x 200 ml) 1) 200 g Dextrose into 200 ml heated diH2O (heat in microwave for 1 min) 2) Stir for awhile 3) Bring to 400 ml 4) Stir until clear 5) Filter sterilize into bottles 80% Glycerol Glycerol from stock shelf 1) Microwave entire bottle 1 min 2) 8 parts glycerol to 2 parts diH2O 3) pipet using glass pipettes for volume ~100 ml. Autoclave 20 minutes Luria Broth (LB bacteria media) 10 g Bacto Tryptone (powder) 5 g Bacto Yeast Extract (powder) 5 g NaCl (crystals) 160 µl 50% NaOH solution (very basic- wear gloves) 1) Dissolve into 1 L tap diH2O 2) Autoclave 35 minutes Tris-buffer, Acetic Acid, EDTA (50X TAE 1 liter) 242 g Tris Base (powder) 57 ml HOAc – glacial acetic acid solution (under hood in cabinet) 100 ml 0.5M EDTA (solution) Water to 1 L This makes 50X TAE of pH 8.4. Do not autoclave or filter sterilize